Distinguishing pca3 messenger rna species in benign and malignant prostate tissues

ABSTRACT

This invention concerns the discovery of two distinct PCA3 mRNA sequences. One of these sequences corresponds to a short PCA3 mRNA molecule whereas the other PCA3 RNA molecule is longer as it comprises an additional sequence between exon 3 and exon 4a. The short RNA is associated with prostate cancer whereas the long RNA sequence is associated with a non-malignant state of the prostate. Based on the differential expression levels of these two PCA3 RNA sequences, protocols for the diagnosis of prostate disease are provided. The invention also relates to therapeutic approaches to prostate cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of 13/080,262, now U.S. Pat. No.8,241,848, which is a continuation of U.S. application Ser. No.12/635,917, now U.S. Pat. No. 7,927,806, which is a continuation of U.S.application Ser. No. 12/098,577, now U.S. Pat. No. 7,655,408, which is adivisional of U.S. patent application Ser. No. 09/675,650, now U.S. Pat.No. 7,368,545, which claims priority to U.S. provisional application No.60/156,594 filed Sep. 29, 1999. The patent applications identified aboveare incorporated here by reference in their entirety to providecontinuity of disclosure.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable formentitled “Sequence Listing”, created Jul. 25, 2012, having a size of 4Kb. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to prostate cancer. More specifically, thepresent invention relates to nucleic acid molecules (messenger RNAs)encoded by the gene PCA3; the differential expression of two of theseRNA species in non-malignant and malignant prostatic states; methods forspecifically diagnosing prostate cancer based on the detection of theRNA species related to prostate cancer; therapeutic approaches toprostate cancer implying these two RNA species; nucleic acid moleculesand antibodies having binding affinity for the differentially expressedmRNAs; kits containing nucleic acid probes or antibodies; bioassaysusing the nucleic acid sequences of the differentially expressed mRNAsof the present invention to diagnose, assess or prognose a mammalafflicted with or susceptible to developing prostate cancer; and tobioassays to screen for compounds which modulate the expression of themRNAs of the present invention.

BACKGROUND OF THE INVENTION

Over the last decade, cancer of the prostate has become the mostcommonly diagnosed malignancy among men and the second leading cause ofmale cancer deaths in the western population, following lung cancer(Landis et al., 1998, CA Cancer J. Clin. 48(1):6-29). Of all cancers,the incidence of prostate cancer increases most rapidly with age. Aslongevity among the western population increases, there continues to bea corresponding rise in the number of prostate cancers with an expectedincrease of 60% in this decade alone. Mortality has increased at aslower rate, but overall has doubled in the last 50 years. Although thedisease is typically diagnosed in men over the age of 65, its impact isstill significant in that the average life span of a man who dies fromprostate cancer is reduced by 9-10 years. If discovered, early prostatecancer can now be cured with surgery in approximately 90% of cases.Unfortunately the disease is slowly fatal once the tumor spreads outsizethe area of the gland and forms distant metastases. Early detection ofthe disease, while still confined to the prostate gland, and accuratestaging for the selection of appropriate therapy should improvemortality rates.

Despite many advances in recent years, the precision with which anindividual suffering from prostate cancer can be staged is stillsub-optimal. The main reason for this is that tumor spread beyond theprostate is generally microscopic rather than macroscopic. Digitalrectal examination of the prostate has been the cornerstone for thelocal staging of prostatic cancer for many decades, but it oftentimesunderestimates the extent of the disease. Transrectal ultrasound byitself is only of limited value as a means of prostate cancer staging.Computer tomography and magnetic resonance imaging have generally beendisappointing in the staging of prostate cancer (Kirby, 1997, Prostatecancer and Prostatic Diseases 1:2-10). Recent promising approaches toprostate cancer staging imply the use of biochemical and moleculartechnologies, centered around proteins or their corresponding nucleicacids which are preferentially expressed in prostate cells (Lange, 1997,In “Principles and Practice of Genitourinary Oncology” ed.Lippincott-Raven Publishers, Ch. 41, pp. 417-425). The most notoriousprostate markers are PSA (prostate specific antigen) and PSM (prostatespecific membrane) antigen.

PSA is a secreted glycoprotein encoded by the PSA gene located onchromosome 19. It is expressed under androgen control by glandularepithelial cells of the prostate and secreted into seminal plasma. PSAprotein is normally confined to the prostate but in the case ofprostatic disease such as cancer or BPH (benign prostate hyperplasia),PSA leaks into the blood where it is present in different forms,including one that is and one that is not bound to protein complexes(El-Shirbiny, 1994, Adv. Clin. Chem. 31:99). The measurement of totalPSA serum concentrations is one of the most frequently used andFDA-approved biochemical tests in the screening and management ofprostate cancer patients. Studies to date have suggested that screeningwith PSA, in conjunction with digital rectal exams and transrectalultrasound, increases the detection of early prostate cancers oftenwhile still localized to the gland itself (Brawer et al., 1992, J. Urol.147:841). Serum PSA is also useful for monitoring of patients aftertherapy, especially after surgical prostatectomy. However, total PSAmeasurements also identify a large number of patients with abnormallyelevated levels who are subsequently found to have no prostate cancer.Recently, the concept of measuring the percent free/total PSA ratio wasshown to increase the specificity of prostate cancer screening in menwith PSA between 4 and 10 ng/mL (Letran et al., 1998, J. Urol. 160:426).

The PSM gene encodes a transmembrane glycoprotein expressed byepithelial cells of normal prostate, benign prostate hyperplasia and, toa greater extent, malignant prostatic tissue. Low levels of PSM are alsodetected in some other tissues (Israeli et al., 1994, Cancer Res.54:1807). PSA and PSM have also been targets for molecular approaches toprostate cancer using RT-PCR (reverse transcription-polymerase chainreaction). This very sensitive nucleic acid amplification technology isused to identify cells based on the expression of specific messengerRNAs. It involves preparing RNA samples from tissues or body fluids,reverse transcribing it into cDNA and amplifying specific cDNAs by theuse of primers that target the particular gene of interest. RT-PCRanalyses of blood, lymph nodes and bone marrow from prostate cancerpatients using PSA and PSM have disclosed the extreme sensitivity ofthis approach. However, the clinical value of molecular tests still hasto be confirmed (Verkaik et al., 1997, Urol. Res. 25:373; Gomella etal., 1997, J. Urol. 158:326).

Thus, there remains a need to provide a more sensitive test fordiagnosing prostate cancer. There also remains a need to provide abetter test for the staging of prostate cancer. There also remains aneed to provide a prostate cancer marker which is more specific and morereliable to prostate cancer detection, staging and treatment methods.

The present invention seeks to meet these and other needs.

A new prostate cancer marker, PCA3, was discovered a few years ago bydifferential display analysis intended to highlight genes associatedwith prostate cancer development (PCT application numberPCT/CA98/00346). PCA3 is located on chromosome 9 and composed of fourexons. It encodes at least four different transcripts which aregenerated by alternative splicing and polyadenylation. By RT-PCRanalysis, PCA3 expression was found to be limited to the prostate andabsent in all other tissues, including testis, ovary, breast andbladder. Northern blot analysis showed that PCA3 is highly expressed inthe vast majority of prostate cancers examined (47 out of 50) whereas noor very low expression is detected in benign prostate hyperplasia ornormal prostate cells from the same patients. There is at least 20-foldoverexpression of PCA3 in prostatic carcinomas in comparison to normalor BPH tissues. PCA3 expression seems to increase with tumor grade andis detected in metastatic lesions.

In summary, prostate cancer staging based on specific markers such asPSA and PSM is a very promising avenue for the management of thedisease. The drawback of using PSA or PSM for prostate cancer staging isthat they are expressed in normal as well as in cancerous cells. Inaddition, poorly differentiated tumors may escape diagnosis since theytend to produce significantly less PSA protein than less aggressivetumors. This is the case for 10% of all prostate cancers. PCA3, on theother hand, is differentially expressed in cancerous and normal prostatecells, and its expression does not decrease with increasing tumor grade.PCA3 could therefore be a useful tool which may overcome the drawbacksof PSA and PSM in the diagnosis, staging and treatment of prostatecancer patients.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference, in their entirety.

SUMMARY OF THE INVENTION

The invention concerns the discovery of distinct PCA3 RNAs associatedwith a non-malignant and/or malignant status of the prostate.

The invention also concerns the identification that a balance betweenthe level of these PCA3 mRNAs correlates with the non-malignant or themalignant status of the prostate.

One of these RNAs corresponds to a PCA3 RNA molecule having anadditional sequence of 228 bp (shown in SEQ ID NO:1), inserted betweenexons 3 and 4a, whereas the other lacks the additional sequence (SEQ IDNO:2). The RNA lacking the additional sequence is associated withprostate cancer whereas the RNA comprising same is associated with anon-malignant prostatic state. Based on the differential expression ofthese two PCA3 RNA species, protocols for the diagnosis of prostatedisease are provided. The above findings could also lead to atherapeutic approach to prostate cancer.

The invention further concerns reagents and methods to assess theprostate status in an animal, comprising a quantitative determination ofSEQ ID NO:1 or fragments, or variants thereof with respect to SEQ IDNO:2 or fragments, or variants thereof.

Thus, the present invention relates to the discovery andcharacterization of a novel sequence expressed in PCA3 mRNA, whichenables a determination of the prostate status of an animal, based on adetermination of the relative abundance of two differentially expressedPCA3 mRNAs.

The invention provides, in general, isolated nucleic acid moleculesencoding differentially expressed PCA3 mRNAs and to variants or portionsthereof, retaining their ability to enable a prostate statusdetermination.

The invention further provides purified polypeptides encoded by thedifferentially expressed PCA3 mRNAs of the present invention or anepitope binding portion thereof.

The invention also provides nucleic acids for the specific detection ofthe presence of differentially expressed PCA3 mRNAs associated withprostate cancer or proteins or polypeptides encoded by such mRNAs in asample.

The invention further provides a method of detecting nucleic acidencoding differentially expressed PCA3 mRNAs.

The invention also provides a kit for detecting the presence of nucleicacid encoding differentially-expressed PCA3 mRNAs in a sample.

The invention in addition provides a recombinant nucleic acid moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina host cell and the above-described isolated nucleic acid molecule,variants or fragments thereof, encoding differentially expressed PCA3mRNAs.

The invention also provides a recombinant nucleic acid moleculecomprising a vector and the above-described isolated nucleic acidmolecule encoding differentially expressed PCA3 mRNAs.

The invention further provides an antisense nucleic acid moleculespecific to the differentially expressed PCA3 mRNAs.

The invention also provides a cell that contains an above-describedrecombinant nucleic acid molecule.

The invention further relates to a non-human organism that contains anabove-described recombinant nucleic acid molecule encoding adifferentially expressed PCA3 mRNA. In particular, the invention relatesto a non-human organism containing a recombinant nucleic acid moleculeencoding a PCA3 mRNA having an additional sequence between exon 3 andexon 4a. In a particularly preferred embodiment, this additionalsequence comprises the sequence of SEQ ID NO:1, variants of partsthereof.

The invention also relates to an antibody having binding affinityspecifically to a polypeptide encoded by a differentially expressed PCA3mRNA or an epitope-bearing portion thereof.

The invention further provides a method of detecting differentiallyexpressed PCA3 mRNAs in a sample. As well, it also provides a method ofmeasuring the amount of differentially expressed PCA3 mRNAs in a sample.

The invention further relates to a method of detecting antibodies havingbinding affinity specifically to polypeptides encoded by adifferentially expressed PCA3 mRNA.

In one embodiment, the invention further relates to a diagnostic kitcomprising a first container means containing nucleic acid moleculesspecific for a differentially expressed PCA3 mRNA, and a secondcontainer means containing a probe specific to a differentiallyexpressed PCA3 mRNAs.

In another embodiment, the invention relates to a diagnostic kitcomprising a first container means containing the above-describedantibody, and a second container means containing a conjugate comprisinga binding partner of the monoclonal antibody and a label.

The invention also provides a hybridoma which produces theabove-described monoclonal antibody.

As well, the invention further relates to diagnostic methods for humandisease, in particular, prostate cancer. Preferably, a method ofdiagnosing the presence or predisposition to develop prostate cancer ina patient is provided herein.

The invention also provides methods for therapeutic uses involving allor part of (1) a nucleic acid sequence encoding the differentiallyexpressed PCA3 mRNAs, variants or parts thereof, (2) antisense todifferentially expressed PCA3 mRNA molecules, variants or parts thereof,(3) protein encoded by a differentially expressed PCA3 mRNA, variants orparts thereof, or (4) antibodies to proteins encoded by differentiallyexpressed PCA3 mRNAs.

Further, the invention provides a method to modulate the level ofdifferentially expressed of a first PCA3 mRNA (e.g. the long one) and asecond PCA3 mRNA (e.g. the short one) by expressing one of the first orsecond differentially expressed mRNA. In a preferred embodiment, theinvention provides a modulation of the differentially expressed PCA3mRNAs such that the level of the first PCA3 mRNA is superior to that ofthe second.

Having identified the differential expression of mRNAs as a marker forprostatic state of an animal, and more particularly having shown thatthe presence of the additional sequence, which interrupts the codingsequence of the PCA3 encoded protein, correlates with a non-malignantstate, while the absence of the additional sequence and anon-interruption of the thereby encoded protein, correlates withmalignant cancer, the present invention therefore provides the means tointerrupt the coding sequence of the PCA3 protein, using any means ofgenetic engineering, known to a skilled artisan, and assesses whethersuch an interruption can revert the malignant phenotype.

In order to provide a clear and consistent understanding of terms usedin the present description, a number of definitions are providedhereinbelow.

As used herein, the terminology “non-malignant prostate or status” ismeant to cover a non-cancerous prostatic state. Thus, theseterminologies are meant to include a normal status as well as a benignprostatic status (such as BPH, for example).

Since the differentiating markers between the malignant andnon-malignant prostatic state is at the mRNA and protein level (i.e., anexpressed marker), one of the advantages of the present invention is toenable a determination of the prostatic status in an animal using anumber of available means to the skilled artisan. Non-limiting examplesof such means include nucleic acid probes, antibodies, ligands and PNAs,in easily obtainable cells which express these differentiating markers.A non-limiting example thereof is lymphocytes, thereby enabling adetermination from a simple blood sample.

The term “sample” is used herein broadly to refer to all types ofsamples from an animal in which the differential expression of the shortand/or long PCA3 nucleic acid or protein of the present invention can beanalyzed. Non-limiting examples thereof include biopsies, blood, fineneedle aspirate, urine and bone marrow.

Nucleotide sequences are presented herein by single strand, in the 5′ to3′ direction, from left to right, using the one letter nucleotidesymbols as commonly used in the art and in accordance with therecommendations of the IUPAC-IUB Biochemical Nomenclature Commission.

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Generally,the procedures for cell cultures, infection, molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as for exampleSambrook et al. (1989, Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratories) and Ausubel et al. (1994, Current Protocolsin Molecular Biology, Wiley, New York).

The present description refers to a number of routinely used recombinantDNA (rDNA) technology terms. Nevertheless, definitions of selectedexamples of such rDNA terms are provided for clarity and consistency.

As used herein, “nucleic acid molecule”, refers to a polymer ofnucleotides. Non-limiting examples thereof include DNA (i.e., genomicDNA, cDNA) and RNA molecules (i.e., mRNA). The nucleic acid molecule canbe obtained by cloning techniques or synthesized. DNA can bedouble-stranded or single-stranded (coding strand or non-coding strand[antisense]).

The term “recombinant DNA” as known in the art refers to a DNA moleculeresulting from the joining of DNA segments. This is often referred to asgenetic engineering.

The term “DNA segment”, is used herein, to refer to a DNA moleculecomprising a linear stretch or sequence of nucleotides. This sequencewhen read in accordance with the genetic code, can encode a linearstretch or sequence of amino acids which can be referred to as apolypeptide, protein, protein fragment and the like.

The terminology “amplification pair” refers herein to a pair ofoligonucleotides (oligos) of the present invention, which are selectedto be used together in amplifying a selected nucleic acid sequence byone of a number of types of amplification processes, preferably apolymerase chain reaction. Other types of amplification processesinclude ligase chain reaction, strand displacement amplification, ornucleic acid sequence-based amplification, as explained in greaterdetail below. As commonly known in the art, the oligos are designed tobind to a complementary sequence under selected conditions.

In one particular embodiment, amplification of a nucleic acid samplefrom a patient is amplified under conditions which favor theamplification of the most abundant differentially expressed nucleicacid. In one preferred embodiment, RT-PCR is carried out on an mRNAsample from a patient under conditions which favor the amplification ofthe most abundant PCA3 mRNA. In another preferred embodiment, theamplification of the differentially expressed PCA3 nucleic acids iscarried out simultaneously. Of course, it will be realized by a personskilled in the art that such methods could be adapted for the detectionof differentially expressed proteins instead of differentially expressednucleic acid sequences.

The nucleic acid (i.e., DNA or RNA) for practicing the present inventionmay be obtained according to well known methods.

Oligonucleotide probes or primers of the present invention may be of anysuitable length, depending on the particular assay format and theparticular needs and targeted genomes employed. In general, theoligonucleotide probes or primers are at least 12 nucleotides in length,preferably between 15 and 24 molecules, and they may be adapted to beespecially suited to a chosen nucleic acid amplification system. Ascommonly known in the art, the oligonucleotide probes and primers can bedesigned by taking into consideration the melting point of hydrizidationthereof with its targeted sequence (see below and in Sambrook et al.,1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, CSHLaboratories; Ausubel et al., 1989, in Current Protocols in MolecularBiology, John Wiley & Sons Inc., N.Y.).

The term “oligonucleotide” or “DNA” molecule or sequence refers to amolecule comprised of the deoxyribonucleotides adenine (A), guanine (G),thymine (T) and/or cytosine (C). When in a double-stranded form, it cancomprise or include a “regulatory element” according to the presentinvention, as the term is defined herein. The term “oligonucleotide” or“DNA” can be found in linear DNA molecules or fragments, viruses,plasmids, vectors, chromosomes or synthetically derived DNA. As usedherein, particular double-stranded DNA sequences may be describedaccording to the normal convention of giving only the sequence in the 5′to 3′ direction. It will also be recognized that “oligonucleotide” canbe in a single-stranded form.

“Nucleic acid hybridization” refers generally to the hybridization oftwo single-stranded nucleic acid molecules having complementary basesequences, which under appropriate conditions will form athermodynamically favored double-stranded structure. Examples ofhybridization conditions can be found in the two laboratory manualsreferred above (Sambrook et al., 1989, supra and Ausubel et al., 1989,supra) and are commonly known in the art. In the case of a hybridizationto a nitrocellulose filter, as for example in the well known Southernblotting procedure, a nitrocellulose filter can be incubated overnightat 65° C. with a labeled probe in a solution containing 50% formamide,high salt (5×SSC or 5×SSPE), 5×Denhardt's solution, 1% SDS, and 100μg/ml denatured carrier DNA (i.e. salmon sperm DNA). Thenon-specifically binding probe can then be washed off the filter byseveral washes in 0.2×SSC/0.1% SDS at a temperature which is selected inview of the desired stringency: room temperature (low stringency), 42°C. (moderate stringency) or 65° C. (high stringency). The selectedtemperature is based on the melting temperature (Tm) of the DNA hybrid.Of course, RNA-DNA hybrids can also be formed and detected. In suchcases, the conditions of hybridization and washing can be adaptedaccording to well known methods by the person of ordinary skill.Stringent conditions will be preferably used (Sambrook et al., 1989,supra).

Probes of the invention can be utilized with naturally occurringsugar-phosphate backbones as well as modified backbones includingphosphorothioates, dithionates, alkyl phosphonates and α-nucleotides andthe like. Modified sugar-phosphate backbones are generally taught byMiller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987,Nucleic acid molecule. Acids Res., 14:5019. Probes of the invention canbe constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid(DNA), and preferably of DNA.

The types of detection methods in which probes can be used includeSouthern blots (DNA detection), dot or slot blots (DNA, RNA), andNorthern blots (RNA detection). Although less preferred, labeledproteins could also be used to detect a particular nucleic acid sequenceto which it binds. More recently, PNAs have been described (Nielsen etal. 1999, Current Opin. Biotechnol. 10:71-75). PNAs could also be usedto detect the mRNAs of the present invention. Other detection methodsinclude kits containing probes on a dipstick setup and the like.

Although the present invention is not specifically dependent on the useof a label for the detection of a particular nucleic acid sequence, sucha label might be beneficial, by increasing the sensitivity of thedetection. Furthermore, it enables automation. Probes can be labeledaccording to numerous well known methods (Sambrook et al., 1989, supra).Non-limiting examples of labels include ³H, ¹⁴C, ³²P, and ³⁵S.Non-limiting examples of detectable markers include ligands,fluorophores, chemiluminescent agents, enzymes, and antibodies. Otherdetectable markers for use with probes, which can enable an increase insensitivity of the method of the invention, include biotin andradionucleotides. It will become evident to the person of ordinary skillthat the choice of a particular label dictates the manner in which it isbound to the probe.

As commonly known, radioactive nucleotides can be incorporated intoprobes of the invention by several methods. Non-limiting examplesthereof include kinasing the 5′ ends of the probes using gamma ³²P ATPand polynucleotide kinase, using the Klenow fragment of Pol I of E. coliin the presence of radioactive dNTP (i.e., uniformly labeled DNA probeusing random oligonucleotide primers in low-melt gels), using the SP6/T7system to transcribe a DNA segment in the presence of one or moreradioactive NTP, and the like.

As used herein, “oligonucleotides” or “oligos” define a molecule havingtwo or more nucleotides (ribo or deoxyribonucleotides). The size of theoligo will be dictated by the particular situation and ultimately on theparticular use thereof and adapted accordingly by the person of ordinaryskill. An oligonucleotide can be synthetised chemically or derived bycloning according to well known methods.

As used herein, a “primer” defines an oligonucleotide which is capableof annealing to a target sequence, thereby creating a double strandedregion which can serve as an initiation point for DNA synthesis undersuitable conditions.

Amplification of a selected, or target, nucleic acid sequence may becarried out by a number of suitable methods. See generally Kwoh et al.,1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniqueshave been described and can be readily adapted to suit particular needsof a person of ordinary skill. Non-limiting examples of amplificationtechniques include polymerase chain reaction (PCR), ligase chainreaction (LCR), strand displacement amplification (SDA),transcription-based amplification, the Qβ replicase system and NASBA(Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi etal., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably,amplification will be carried out using PCR.

Polymerase chain reaction (PCR) is carried out in accordance with knowntechniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188 (the disclosures of all three U.S. patent are incorporatedherein by reference). In general, PCR involves, a treatment of a nucleicacid sample (e.g., in the presence of a heat stable DNA polymerase)under hybridizing conditions, with one oligonucleotide primer for eachstrand of the specific sequence to be detected. An extension product ofeach primer which is synthesized is complementary to each of the twonucleic acid strands, with the primers sufficiently complementary toeach strand of the specific sequence to hybridize therewith. Theextension product synthesized from each primer can also serve as atemplate for further synthesis of extension products using the sameprimers. Following a sufficient number of rounds of synthesis ofextension products, the sample is analysed to assess whether thesequence or sequences to be detected are present. Detection of theamplified sequence may be carried out by visualization following EtBrstaining of the DNA following gel electrophoresis, or using a detectablelabel in accordance with known techniques, and the like. For a review onPCR techniques (see PCR Protocols, A Guide to Methods andAmplifications, Michael et al. Eds, Acad. Press, 1990).

Ligase chain reaction (LCR) is carried out in accordance with knowntechniques (Weiss, 1991, Science 254:1292). Adaptation of the protocolto meet the desired needs can be carried out by a person of ordinaryskill. Strand displacement amplification (SDA) is also carried out inaccordance with known techniques or adaptations thereof to meet theparticular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).

As used herein, the term “gene” is well known in the art and relates toa nucleic acid sequence defining a single protein or polypeptide. A“structural gene” defines a DNA sequence which is transcribed into RNAand translated into a protein having a specific amino acid sequencethereby giving rise the a specific polypeptide or protein. It will bereadily recognized by the person of ordinary skill, that the nucleicacid sequence of the present invention can be incorporated into anyoneof numerous established kit formats which are well known in the art.

A “heterologous” (i.e., a heterologous gene) region of a DNA molecule isa subsegment segment of DNA within a larger segment that is not found inassociation therewith in nature. The term “heterologous” can besimilarly used to define two polypeptidic segments not joined togetherin nature. Non-limiting examples of heterologous genes include reportergenes such as luciferase, chloramphenicol acetyl transferase,8-galactosidase, and the like which can be juxtaposed or joined toheterologous control regions or to heterologous polypeptides.

The term “vector” is commonly known in the art and defines a plasmidDNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicleinto which DNA of the present invention can be cloned. Numerous types ofvectors exist and are well known in the art.

The term “expression” defines the process by which a gene is transcribedinto mRNA (transcription), the mRNA is then being translated(translation) into one polypeptide (or protein) or more.

The terminology “expression vector” defines a vector or vehicle asdescribed above but designed to enable the expression of an insertedsequence following transformation into a host. The cloned gene (insertedsequence) is usually placed under the control of control elementsequences such as promoter sequences. The placing of a cloned gene undersuch control sequences is often referred to as being operably linked tocontrol elements or sequences.

Operably linked sequences may also include two segments that aretranscribed onto the same RNA transcript. Thus, two sequences, such as apromoter and a “reporter sequence” are operably linked if transcriptioncommencing in the promoter will produce an RNA transcript of thereporter sequence. In order to be “operably linked” it is not necessarythat two sequences be immediately adjacent to one another.

Expression control sequences will vary depending on whether the vectoris designed to express the operably linked gene in a prokaryotic oreukaryotic host or both (shuttle vectors) and can additionally containtranscriptional elements such as enhancer elements, terminationsequences, tissue-specificity elements, and/or translational initiationand termination sites.

Prokaryotic expressions are useful for the preparation of largequantities of the protein encoded by the DNA sequence of interest. Thisprotein can be purified according to standard protocols that takeadvantage of the intrinsic properties thereof, such as size and charge(i.e., SDS gel electrophoresis, gel filtration, centrifugation, ionexchange chromatography . . . ). In addition, the protein of interestcan be purified via affinity chromatography using polyclonal ormonoclonal antibodies. The purified protein can be used for therapeuticapplications.

The DNA construct can be a vector comprising a promoter that is operablylinked to an oligonucleotide sequence of the present invention, which isin turn, operably linked to a heterologous gene, such as the gene forthe luciferase reporter molecule. “Promoter” refers to a DNA regulatoryregion capable of binding directly or indirectly to RNA polymerase in acell and initiating transcription of a downstream (3′ direction) codingsequence. For purposes of the present invention, the promoter is boundat its 3′ terminus by the transcription initiation site and extendsupstream (5′ direction) to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter will be found a transcription initiationsite (conveniently defined by mapping with S1 nuclease), as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “COAT” boxes. Prokaryotic promoterscontain −10 and −35 consensus sequences, which serve to initiatetranscription and the transcript products contain a Shine-Dalgarnosequence, which serves a ribosome binding sequences during translationinitiation.

As used herein, the designation “functional derivative” denotes, in thecontext of a functional derivative of a sequence whether an nucleic acidor amino acid sequence, a molecule that retains a biological activity(either function or structural) that is substantially similar to that ofthe original sequence. This functional derivative or equivalent may be anatural derivative or may be prepared synthetically. Such derivativesinclude amino acid sequences having substitutions, deletions, oradditions of one or more amino acids, provided that the biologicalactivity of the protein is conserved. The same applies to derivatives ofnucleic acid sequences which can have substitutions, deletions, oradditions of one or more nucleotides, provided that the biologicalactivity of the sequence is generally maintained. When relating to aprotein sequence, the substituting amino acid as chemico-physicalproperties which are similar to that of the substituted amino acid. Thesimilar chemico-physical properties include, similarities in charge,bulkiness, hydrophobicity, hydrophylicity and the like. The term“functional derivatives” is intended to include “fragments”, “segments”,“variants”, “analogs” or “chemical derivatives” of the subject matter ofthe present invention.

Thus, the term “variant” refers herein to a protein or nucleic acidmolecule which is substantially similar in structure and biologicalactivity to the protein or nucleic acid of the present invention.

The functional derivatives of the present invention can be synthesizedchemically or produced through recombinant DNA technology. All thesemethods are well known in the art.

As used herein, “chemical derivatives” is meant to cover additionalchemical moieties not normally part of the subject matter of theinvention. Such moieties could affect the physico-chemicalcharacteristic of the derivative (i.e., solubility, absorption, halflife and the like, decrease of toxicity). Such moieties are exemplifiedin Remington's Pharmaceutical Sciences (1980). Methods of coupling thesechemical-physical moieties to a polypeptide are well known in the art.

The term “allele” defines an alternative form of a gene which occupies agiven locus on a chromosome.

As commonly known, a “mutation” is a detectable change in the geneticmaterial which can be transmitted to a daughter cell. As well known, amutation can be, for example, a detectable change in one or moredeoxyribonucleotide. For example, nucleotides can be added, deleted,substituted for, inverted, or transposed to a new position. Spontaneousmutations and experimentally induced mutations exist. The result of amutations of nucleic acid molecule is a mutant nucleic acid molecule. Amutant polypeptide can be encoded from this mutant nucleic acidmolecule.

As used herein, the term purified refers to a molecule having beenseparated from a cellular component. Thus, for example, a purifiedprotein has been purified to a level not found in nature. Asubstantially pure molecule is a molecule that is lacking in all othercellular components.

As used herein, the terms molecule, compound, or agent are usedinterchangeably and broadly to refer to natural, synthetic orsemi-synthetic molecules or compounds. The term molecule thereforedenotes for example chemicals, macromolecules, cell or tissue extracts(from plants or animals) and the like. Non limiting examples ofmolecules include nucleic acid molecules, peptides, ligands, includingantibodies, carbohydrates and pharmaceutical agents. The agents can beselected and screened by a variety of means including random screening,rational selection and by rational design using for example protein orligand modelling methods such as computer modelling. The termsrationally selected or rationally designed are meant to define compoundswhich have been chosen based on the configuration of the interactiondomains of the present invention. As will be understood by the person ofordinary skill, macromolecules having non-naturally occurringmodifications are also within the scope of the term molecule. Forexample, peptidomimetics, well known in the pharmaceutical industry andgenerally referred to as peptide analogs can be generated by modellingas mentioned above. Similarly, in a preferred embodiment, thepolypeptides of the present invention are modified to enhance theirstability. It should be understood that in most cases this modificationshould not alter the biological activity of the protein. The moleculesidentified in accordance with the teachings of the present inventionhave a therapeutic value in diseases or conditions in which thephysiology or homeostasis of the cell and/or tissue is compromised by adefect in the expression of PCA3 mRNAs. Alternatively, the moleculesidentified in accordance with the teachings of the present inventionfind utility in the development of compounds which can modulate theexpression of a differentially expressed PCA3 mRNA or modulate theactivity or level of a protein encoded thereby.

As used herein, agonists and antagonists also include potentiators ofknown compounds with such agonist or antagonist properties. In oneembodiment, modulators of the level or the activity of the PCA3 proteinlacking the additional sequence of the present invention can beidentified and selected by contacting the indicator cell with a compoundor mixture or library of molecules for a fixed period of time. Incertain embodiments, the additional sequence-containing PCA3 protein canserve as a control.

The present invention also provides antisense nucleic acid moleculeswhich can be used for example to decrease or abrogate the expression ofthe PCA3 mRNA lacking the additional sequence of the present inventionor of the protein encoded thereby. An antisense nucleic acid moleculeaccording to the present invention refers to a molecule capable offorming a stable duplex or triplex with a portion of its targetednucleic acid sequence (DNA or RNA). The use of antisense nucleic acidmolecules and the design and modification of such molecules is wellknown in the art as described for example in WO 96/32966, WO 96/11266,WO 94/15646, WO 93/08845 and U.S. Pat. No. 5,593,974. Antisense nucleicacid molecules according to the present invention can be derived fromthe nucleic acid sequences and modified in accordance to well knownmethods. For example, some antisense molecules can be designed to bemore resistant to degradation to increase their affinity to theirtargeted sequence, to affect their transport to chosen cell types orcell compartments, and/or to enhance their lipid solubility by usingnucleotide analogs and/or substituting chosen chemical fragmentsthereof, as commonly known in the art.

An indicator cell in accordance with the present invention can be usedto identify antagonists. For example, the test molecule or molecules areincubated with the host cell in conjunction with one or more agonistsheld at a fixed concentration. An indication and relative strength ofthe antagonistic properties of the molecule(s) can be provided bycomparing the level of gene expression in the indicator cell in thepresence of the agonist, in the absence of test molecules vs in thepresence thereof. Of course, the antagonistic effect of a molecule canalso be determined in the absence of agonist, simply by comparing thelevel of expression of the reporter gene product in the presence andabsence of the test molecule(s).

It shall be understood that the “in vivo” experimental model can also beused to carry out an “in vitro” assay. For example, cellular extractsfrom the indicator cells can be prepared and used in one of theaforementioned “in vitro” tests.

As used herein the recitation indicator cells refers to cells thatexpress a differentially expressed PCA3 mRNA according to the presentinvention. In some embodiment, the protein encoded by the nucleic acidsequence can be coupled to an identifiable or selectable phenotype orcharacteristic. Such indicator cells can be used in the screening assaysof the present invention. In certain embodiments, the indicator cellshave been engineered so as to express a chosen derivative, fragment,homolog, or mutant of the differentially-expressed PCA3 mRNA of thepresent invention. The cells can be yeast cells or higher eukaryoticcells such as mammalian cells. When the binding partner for the PCA3protein will have been identified, the interaction between the twopartners will be able to serve as a target for the modulation of theactivity of this PCA3-encoded protein. In one particular embodiment, theindicator cell would be a yeast cell harboring vectors enabling the useof the two hybrid system technology, as well known in the art (Ausubelet al., 1994, supra) and can be used to test a compound or a librarythereof. In one embodiment, a reporter gene encoding a selectable markeror an assayable protein can be operably linked to a control element suchthat expression of the selectable marker or assayable protein isdependent on the interaction of the PCA3-encoded protein and its bindingpartner. Such an indicator cell could be used to rapidly screen athigh-throughput a vast array of test molecules. In a particularembodiment, the reporter gene is luciferase or β-Gal.

In some embodiments, it might be beneficial to express a protein of thepresent invention as a fusion protein. The design of constructs thereforand the expression and production of fusion proteins and are well knownin the art (Sambrook et al., 1989, supra; and Ausubel et al., 1994,supra).

Non limiting examples of such fusion proteins include a hemaglutininfusions and Gluthione-5-transferase (GST) fusions and Maltose bindingprotein (MBP) fusions. In certain embodiments, it might be beneficial tointroduce a protease cleavage site between the two polypeptide sequenceswhich have been fused. Such protease cleavage sites between twoheterologously fused polypeptides are well known in the art.

In certain embodiments, it might also be beneficial to fuse the proteinof the present invention to signal peptide sequences enabling asecretion of the fusion protein from the host cell. Signal peptides fromdiverse organisms are well known in the art. Bacterial OmpA and yeastSuc2 are two non limiting examples of proteins containing signalsequences. In certain embodiments, it might also be beneficial tointroduce a linker (commonly known) between the interaction domain andthe heterologous polypeptide portion. Such fusion protein find utilityin the assays of the present invention as well as for purificationpurposes, detection purposes and the like.

For certainty, the sequences and polypeptides useful to practice theinvention include without being limited thereto mutants, homologs,subtypes, alleles and the like. It shall be understood that generally,the sequences of the present invention should encode a functional(albeit defective) PCA3 protein. It will be clear to the person ofordinary skill that whether the PCA3 of the present invention, variant,derivative, or fragment thereof retains its can be determined by usingthe teachings and assays of the present invention and the generalteachings of the art.

As exemplified herein below, the PCA3 protein of the present inventioncan be modified, for example by in vitro mutagenesis, to dissect thestructure-function relationship thereof and permit a better design andidentification of modulating compounds. However, some derivative oranalogs having lost their biological function may still find utility,for example for raising antibodies. These antibodies could be used fordetection or purification purposes. In addition, these antibodies couldalso act as competitive or non-competitive inhibitor and be found to bemodulators of the activity of the PCA3 protein of the present invention.

A host cell or indicator cell has been “transfected by exogenous orheterologous DNA (e.g., a DNA construct) when such DNA has beenintroduced inside the cell. The transfecting DNA may or may not beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell. In prokaryotes, yeast, and mammalian cells for example, thetransfecting DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transfected cell isone in which the transfecting DNA has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transfecting DNA.Transfection methods are well known in the art (Sambrook et al., 1989,supra; Ausubel et al., 1994 supra). The use of a mammalian cell asindicator can provide the advantage of furnishing an intermediatefactor, which permits for example the interaction of two polypeptideswhich are tested, that might not be present in lower eukaryotes orprokaryotes. It will be understood that extracts from mammalian cellsfor example could be used in certain embodiments, to compensate for thelack of certain factors.

In general, techniques for preparing antibodies (including monoclonalantibodies and hybridomas) and for detecting antigens using antibodiesare well known in the art (Campbell, 1984, In Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow etal., 1988 (in: Antibody-A Laboratory Manual, CSH Laboratories). Thepresent invention also provides polyclonal, monoclonal antibodies, orhumanized versions thereof, chimeric antibodies and the like whichinhibit or neutralize their respective interaction domains and/or arespecific thereto.

From the specification and appended claims, the term therapeutic agentshould be taken in a broad sense so as to also include a combination ofat least two such therapeutic agents. Further, the DNA segments orproteins according to the present invention can be introduced intoindividuals in a number of ways. For example, prostatic cells can beisolated from the afflicted individual, transformed with a DNA constructaccording to the invention and reintroduced to the afflicted individualin a number of ways. Alternatively, the DNA construct can beadministered directly to the afflicted individual. The DNA construct canalso be delivered through a vehicle such as a liposome, which can bedesigned to be targeted to a specific cell type, and engineered to beadministered through different routes.

For administration to humans, the prescribing medical professional willultimately determine the appropriate form and dosage for a givenpatient, and this can be expected to vary according to the chosentherapeutic regimen (i.e., DNA construct, protein, cells), the responseand condition of the patient as well as the severity of the disease.

Composition within the scope of the present invention should contain theactive agent (i.e. fusion protein, nucleic acid, and molecule) in anamount effective to achieve the desired therapeutic effect whileavoiding adverse side effects. Typically, the nucleic acids inaccordance with the present invention can be administered to mammals(i.e., humans) in doses ranging from 0.005 to 1 mg per kg of body weightper day of the mammal which is treated. Pharmaceutically acceptablepreparations and salts of the active agent are within the scope of thepresent invention and are well known in the art (Remington'sPharmaceutical Science, 16th Ed., Mack Ed.). For the administration ofpolypeptides, antagonists, agonists and the like, the amountadministered should be chosen so as to avoid adverse side effects. Thedosage will be adapted by the clinician in accordance with conventionalfactors such as the extent of the disease and different parameters fromthe patient. Typically, 0.001 to 50 mg/kg/day will be administered tothe mammal.

The present invention relates to a kit for diagnosing and/or stagingprostate cancer or a predisposition to contracting same comprising anucleic acid, a protein or a ligand in accordance with the presentinvention. For example, a compartmentalized kit in accordance with thepresent invention includes any kit in which reagents are contained inseparate containers. Such containers include small glass containers,plastic containers or strips of plastic or paper. Such containers allowthe efficient transfer of reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample(DNA, protein or cells), a container which contains the primers used inthe assay, containers which contain enzymes, containers which containwash reagents, and containers which contain the reagents used to detectthe extension products.

The present invention also relates to a kit comprising theoligonucleotide primer of the present invention, which are specific toeither one of the PCA3 mRNA lacking the additional sequence of thepresent invention or the PCA3 mRNA containing the additional sequence ofthe present invention.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the invention, reference will now bemade to the accompanying drawings, showing by way of illustration apreferred embodiment thereof, and in which:

FIG. 1 shows the PCA3 genomic structure and location of oligonucleotidesused for PCR;

FIG. 2 shows a gel separating PCA3 RT-PCR products amplified from tissuebiopsies of prostate cancer and benign prostate hyperplasia, using theprimers of Example 1;

FIG. 3 illustrates the nucleic acid sequences of RT-PCR-amplified PCA3fragments with and without the additional sequence of the presentinvention. The sequences were amplified using PCR primers located inexon 3 and exon 4a. Primer sequences are shown in bold letters. Capitalletters represent nucleic acids common to both sequences;

FIG. 4 shows the amino acid sequence predicted from the PCA3 mRNAscontaining the additional sequence of the present invention. Thissequence corresponds to amino acids 1-23 of the original PCA3polypeptide; and

FIG. 5 shows examples of antigenic epitope-bearing PCA3 peptidescomprising 8 amino acids (calculated according to H. G. Rammensee etal., 1995, MHC ligands and peptide motifs: first listing, inImmunogenics; 41(4)). The SEQ ID NOs of the exemplified antigenicepitopes are indicated on the right (SEQ ID NOs 5 to 12).

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments with reference to the accompanyingdrawing which is exemplary and should not be interpreted as limiting thescope of the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one embodiment, the present invention relates to an isolated and/orpurified differentially-expressed PCA3 mRNA molecule. Preferably, thePCA3 mRNA or nucleic acid molecule comprises a polynucleotide sequenceat least 90% identical (more preferably, 95%, 96%, 97%, 98%, 99% or 100%identical) to a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a differentially expressed PCA3polypeptide comprising the complete amino acid sequence in SEQ ID NO: 3;(b) a nucleotide sequence complementary to any of the nucleotidesequences in (a) or (b).

In another embodiment, the isolated nucleic acid molecule comprises adifferentially expressed PCA3 mRNA nucleotide sequence with greater than90% identity or similarity to the nucleotide sequence present in SEQ IDNO: 1 (preferably greater than 95%, 96%, 97%, 98%, 99% or 100%). Inanother preferred embodiment, the isolated nucleic acid moleculecomprises the differentially expressed PCA3 mRNA sequence lacking theadditional sequence present in SEQ ID NO:2. In another embodiment, theisolated differentially expressed additional sequence-minus mRNAsequence nucleic acid molecule encodes the differentially expressed PCA3amino acid sequence present in SEQ ID NO:3.

Although PCT application CA98/00346 teaches a number of alternativelyspliced mRNAs, prior to the present invention, a PCA3 mRNA comprising anadditional sequence between exon 3 and exon 4a had not been identified.Furthermore, the identification of this additional sequence as adistinguishing marker of the prostate state had not been made. Inaddition, the correlation between the PCA3 mRNA minus the additionalsequence and prostate cancer (as opposed to the PCA3 mRNA-containing theadditional sequence in non-prostate cancer [i.e., normal or BPH]) hadnot been made. Thus, the additional sequence in PCA3 mRNA enables aprognosis and diagnosis of prostatic diseases in a patient. Preferably,the PCA3 nucleic acid molecule comprises a polynucleotide sequence atleast 90% identical (more preferably, 95%, 96%, 97%, 98%, 99% or 100%identical) to one of the above-described differentially expressed mRNAs.

Also included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules andderivatives thereof. For example, the nucleic acid sequences depicted inSEQ ID NO:1 and SEQ ID NO:2 can be altered by substitutions, additionsor deletions that provide for functionally equivalent molecules. Due tothe degeneracy of nucleotide coding sequences, other DNA sequences whichencode substantially the same amino acid sequence as depicted in SEQ IDNO: 3 can be used in the practice of the present invention.

In addition, the nucleic acid sequence can comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in SEQ ID NO:1 or 2 or a derivative thereof. Anynucleotide or polynucleotide can be used in this regard, provided thatits addition, deletion or substitution does not substantially alter theamino acid sequence of SEQ ID NO:3 which is encoded by the additionalsequence-containing nucleotide sequence. Moreover, the nucleic acidmolecule of the present invention can, as necessary, have restrictionendonuclease recognition sites added to its 5′-end and/or 3′-end. Allvariations of the nucleotide sequence of the PCA3 nucleotide codingsequence and fragments thereof permitted by the genetic code are,therefore, included in this invention.

Further, it is possible to delete codons or to substitute one or morecodons by codons other than degenerate codons to produce a structurallymodified polypeptide, but one which has substantially the same utilityor activity of the polypeptide produced by the unmodified nucleic acidmolecule. As recognized in the art, the two polypeptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

One skilled in the art will realize that genomes often contain slightallelic variations between individuals. Therefore, the isolated nucleicacid molecule is also intended to include allelic variations, so long asthe sequence is a functional derivative of the differentially expressedPCA3 mRNA coding sequence. When a PCA3 allele does not encode theidentical sequence to that found in SEQ ID Nos:1 or 2, it can beisolated and identified as PCA3 using the same techniques used herein,and especially PCR techniques to amplify the appropriate gene withprimers based on the sequences disclosed herein.

One skilled in the art will realize that organisms other than humansmight also contain differentially-expressed PCA3 mRNAs (for example,eukaryotes; more specifically, mammals, birds, fish, and plants; morespecifically, gorillas, rhesus monkeys, and chimpanzees). The inventionis intended to include, but not be limited to, differentially-expressedPCA3 mRNAs isolated from the above-described organisms.

Isolated nucleic acid molecules of the present invention are also meantto include those chemically synthesized. For example, a nucleic acidmolecule with the nucleotide sequence described herein or encoding theherein described differentially expressed products of PCA3 gene can bedesigned and, if necessary, divided into appropriate smaller fragments.Then an oligomer which corresponds to the nucleic acid molecule, or toeach of the divided fragments, can be synthesized. Such syntheticoligonucleotides can be prepared, for example, by the triester method ofMatteucci et al., J. Am. Chem. Soc. 103:3185-3191 (1981) or by using anautomated DNA synthesizer.

In another embodiment, the present invention relates to purifieddifferentially expressed polypeptides (preferably, substantially pure)having an amino acid sequence corresponding to the herein describedPCA3, or a functional derivative thereof. In a preferred embodiment, thepolypeptide has the amino acid sequence set forth in SEQ ID NO: 3 ormutant or species variation thereof, or at least 80% identity or atleast 90% similarity thereof (preferably, at least 90%, 95%, 96%, 97%,98%, or 99% identity or at least 95%, 96%, 97%, 98%, or 99% similaritythereof), or at least 6 contiguous amino acids thereof (preferably, atleast 10, 15, 20, 25, or 50 contiguous amino acids thereof).

In an additional embodiment, the invention relates todifferentially-expressed PCA3 epitopes. The epitope of thesepolypeptides is an immunogenic or antigenic epitope. An immunogenicepitope is that part of the protein which elicits an antibody responsewhen the whole protein is the immunogen. An antigenic epitope is afragment of the protein which can elicit an antibody response. Methodsof selecting antigenic epitope fragments are well known in the art. See,Sutcliffe et al., Science 219:660-666 (1983). Antigenic epitope-bearingpeptides and polypeptides of the invention are useful to raise an immuneresponse that specifically recognizes the polypeptides. Antigenicepitope-bearing peptides and polypeptides of the invention comprise atleast 7 amino acids (preferably, 9, 10, 12, 15 or 20 amino acids) of theproteins of the invention. Examples of an antigenic peptide are shown inFIG. 5 as predicted using the method of Rammensee et al., (1995, MHCligands and peptide motifs: first listing, in Immunogenics; 41(4)). Ofcourse, it will be realized that other epitope-bearing PCA3 peptidescould be predicted and used to raise antibodies.

A variety of methodologies known in the art can be utilized to obtainthe peptide of the present invention. In one embodiment, the peptide ispurified from tissues or cells which naturally produce the peptide.Alternatively, the above-described isolated nucleic acid fragments canbe used to express the differentially expressed PCA3 protein in anyorganism. The samples of the present invention include cells, proteinextracts or membrane extracts of cells, or biological fluids. The samplewill vary based on the assay format, the detection method and the natureof the tissues, cells or extracts used as the sample.

Any organism can be used as a source for the peptide of the invention,as long as the source organism naturally contains such a peptide. Asused herein, “source organism” refers to the original organism fromwhich the amino acid sequence of the subunit is derived, regardless ofthe organism the subunit is expressed in and ultimately isolated from.

In another embodiment, the present invention relates to a nucleic acidfor the specific detection of the presence of PCA3 nucleic acid in asample comprising the above-described nucleic acid molecules or at leasta fragment thereof which binds under stringent conditions to PCA3nucleic acid.

In yet another embodiment, the present invention relates to nucleic acidprobes which are complementary to a nucleotide sequence consisting of atleast 10 consecutive nucleotides (preferably, 15, 18, 20, 25, or 30)from the nucleic acid molecule comprising a polynucleotide sequence atleast 90% identical to a sequence selected from the group consisting of:

-   -   (a) a nucleotide sequence encoding the PCA3 polypeptide        comprising the complete amino acid sequence in SEQ ID NO: 3;    -   (b) a nucleotide sequence encoding the PCA3 gene comprising the        nucleotide sequence in SEQ ID NO: 1 or 2;    -   (c) a nucleotide sequence complementary to any of the nucleotide        sequences in (a) or (b), and    -   (d) a nucleotide sequence as previously described above.

In one embodiment of the above described method, a nucleic acid probe isimmobilized on a solid support. Examples of such solid supports include,but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, and acrylic resins, such aspolyacrylamide and latex beads. Techniques for coupling nucleic acidprobes to such solid supports are well known in the art.

The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

In another embodiment, the present invention relates to a method ofdetecting the presence of differentially expressed PCA3 mRNA in a samplecomprising: a) contacting the sample with the above-described nucleicacid probe, under specific hybridization conditions such thathybridization occurs, and b) detecting the presence of the probe boundto the nucleic acid molecule. One skilled in the art would select thenucleic acid probe according to techniques known in the art as describedabove. Samples to be tested include but should not be limited to RNAsamples from human tissue.

Having identified that the additional PCA3 sequence of the presentinvention can be used as a marker for distinguishing between malignantand non malignant prostate states, probes which are specific to thisadditional sequence (or variants or fragments thereof) could also beused in accordance with the present invention. Of course, since incertain embodiments, such probes might detect genomic DNA, a positivesignal coming from the genomic DNA might have to be eliminated in orderto specifically detect the differentially expressed PCA3 mRNA.

Although the present invention is specifically demonstrated usingprimers hybridizing to exon 3 and exon 4a sequences, it should be clearto the skilled artisan that primers derived from other regions of PCA3could be used. For example, primers could be derived from sequences ofexon 2, exon 4b, exon 4c or to the additional sequence thereof. Methodsto derive specific primers from known sequences are well known in theart.

In another embodiment, the present invention relates to a kit fordetecting the presence of differentially expressed PCA3 mRNA in a samplecomprising at least one container means having disposed therein theabove-described nucleic acid probe. In a preferred embodiment, the kitfurther comprises other containers comprising one or more of thefollowing: wash reagents and reagents capable of detecting the presenceof bound nucleic acid probe. Examples of detection reagents include, butare not limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes(biotin, avidin, or streptavidin).

In more detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow the efficient transfer of reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the testsample, a container which contains the probe or primers used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, and the like), and containers whichcontain the reagents used to detect the hybridized probe, boundantibody, amplified product, or the like.

One skilled in the art will readily recognize that the nucleic acidprobes described in the present invention can readily be incorporatedinto one of the established kit formats which are well known in the art.

In another embodiment of the present invention (and similarly to probesof the present invention) the antibodies of the present invention can beimmobilized on a solid support. Examples of such solid supports includeplastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, acrylic resins and such as polyacrylamide and latexbeads. Techniques for coupling antibodies to such solid supports arewell known in the art. The immobilized antibodies of the presentinvention can be used for in vitro, in vivo, and in situ assays as wellas in immunochromatography.

In another embodiment, the present invention relates to a method ofdetecting a differentially expressed PCA3 polypeptide in a sample,comprising: a) contacting the sample with an above-described antibody(or protein), under conditions such that immunocomplexes form, and b)detecting the presence of the antibody bound to the polypeptide. Indetail, the methods comprise incubating a test sample with one or moreof the antibodies of the present invention and assaying whether theantibody binds to the test sample. The relative levels of differentiallyexpressed PCA3 in a sample enable a distinction between a malignant andnon-malignant prostatic state.

In a further embodiment, the present invention relates to a method ofdetecting a PCA3 antibody in a sample, comprising: a) contacting thesample with an above-described differentially expressed PCA3 protein,under conditions such that immunocomplexes form, and b) detecting thepresence of the protein bound to the antibody or antibody bound to theprotein. In detail, the methods comprise incubating a test sample withone or more of the proteins of the present invention and assayingwhether the antibody binds to the test sample.

In another embodiment of the present invention, a kit is provided whichcontains all the necessary reagents to carry out the previouslydescribed methods of detection.

The kit can comprise: i) a first container means containing anabove-described antibody, and ii) second container means containing aconjugate comprising a binding partner of the antibody and a label.

The kit can also comprise: i) a first container means containing anabove-described protein, and preferably, ii) second container meanscontaining a conjugate comprising a binding partner of the protein and alabel. More specifically, a diagnostic kit comprises a differentiallyexpressed PCA3 protein as described above, to detect antibodies in theserum of potentially infected animals or humans.

In another preferred embodiment, the kit further comprises one or moreother containers comprising one or more of the following: wash reagentsand reagents capable of detecting the presence of bound antibodies.Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody. The compartmentalizedkit can be as described above for nucleic acid probe kits.

One skilled in the art will readily recognize that the antibodiesdescribed in the present invention can readily be incorporated into oneof the established kit formats which are well known in the art.

It is to be understood that although the following discussion isspecifically directed to human patients, the teachings are alsoapplicable to any animal that differentially expresses PCA3 mRNAs.

The diagnostic and screening methods of the invention are especiallyuseful for a patient suspected of being at risk for developing a diseaseassociated with an altered expression level of PCA3 based on familyhistory, or a patient in which it is desired to diagnose a PCA3-relateddisease (ex. prostate cancer).

According to the invention, presymptomatic screening of an individual inneed of such screening is now possible using DNA encoding the PCA3protein or the PCA3 gene of the invention or fragments thereof. Thescreening method of the invention allows a presymptomatic diagnosis, ofthe presence of the PCA3-minus additional sequence, differentiallyexpressed PCA3 mRNA in individuals, and thus an opinion concerning thelikelihood that such individuals would develop or have developed aPCA3-associated disease or have a normal prostatic state. This isespecially valuable for the identification of carriers of altered PCA3genes, for example, from individuals with a family history of aPCA3-associated disease. Early diagnosis is also desired to maximizeappropriate timely intervention.

In one embodiment of the method of screening, a tissue sample would betaken from such individual, and screened for (1) the presence of thePCA3 mRNA lacking the additional sequence of the present invention; (2)the presence of the additional sequence-containing PCA3 mRNA and/or (3)the presence of differentially expressed PCA3 protein. PCA3 mRNA can becharacterized and compared to determine differentially expressed PCA3mRNA (a) levels and/or (b) size. Lastly, differentially expressed PCA3protein can be (a) detected and/or (b) quantitated using a biologicalassay for PCA3 activity or using an immunological assay and PCA3antibodies A presence of a PCA3 mRNA lacking the additional sequence (oran mRNA not modifying and/or not interrupting the PCA3 coding sequence)and/or of the protein encoded thereby would indicate that the patient isat risk for developing prostate cancer, or has developed prostatecancer. A presence of a PCA3 mRNA containing the additional sequence ofthe present invention and/or of the protein encoded thereby, in theabsence of PCA3 mRNA lacking the additional sequence and/or the proteinencoded thereby or at a level superior to that of the mRNA lacking theadditional sequence and/or the protein encoded thereby would indicatethat the patient has not yet developed prostate cancer, and/or has alower risk of developing prostate cancer.

Therapeutic effects of therapeutic nucleic acids can include, but arenot limited to turning off or modifying the processing of thedifferentially expressed PCA3 mRNA lacking the additional sequence ofthe present invention. In addition, an expression of adifferentially-expressed PCA3 mRNA comprising the additional sequence inaccordance with the present invention to a higher level than that of thePCA3 mRNA lacking the additional sequence could have cancer-reversingeffects on cells.

Included as well in the invention are pharmaceutical compositionscomprising an effective amount of at least one antisense oligonucleotideto a PCA3 mRNA lacking the additional sequence, in combination with apharmaceutically acceptable carrier. Such antisense oligos include, butare not limited to, at least one nucleotide sequence of 12-500 bases inlength which is complementary to at least a portion of SEQ ID NO:2.

Thus, broadly, the invention provides means to shift the balance betweenthe quantity of the differentially expressed PCA3 mRNAs such that themalignant state of a cell can be modulated.

Specificity for gene expression in prostate cancer cells can beconferred by using appropriate cell-specific regulatory sequences, suchas cell-specific enhancers and promoters. Thus, gene therapy can be usedto alleviate PCA3 related pathology by inhibiting the inappropriateexpression of a particular form of PCA3. Moreover, gene therapy can beused to alleviate such pathologies by providing the appropriateexpression level of a particular form of PCA3. In this case, particularPCA3 nucleic acid sequences can be coded by DNA or RNA constructs whichare administered in the form of viruses, as described above.

The present invention provides the above-described PCA3 antibodies(preferably, PCA3 murine antibodies and chimeric PCA3 murine-humanantibodies, and fragments and regions thereof) which inhibit orneutralize PCA3 biological activity in vivo and are specific for PCA3.These antibodies can be used for therapeutic purposes in subjects havingpathologies or conditions associated with the presence of aberrant PCA3expression. Antibodies, and fragments, regions and derivatives thereof,of the present invention preferably contain at least one region whichrecognizes an epitope of PCA3 which has inhibiting and/or neutralizingbiological activity in vivo.

Treatment comprises parenterally administering a single or multipledoses of the antibody, fragment or derivative. Preferred for humanpharmaceutical use are high affinity potent PCA3-inhibiting and/orneutralizing murine and chimeric antibodies, fragments and regions ofthis invention.

Monoclonal antibodies of the present invention may be administered byany means that enables the active agent to reach the agent's site ofaction in the body of a mammal. Because proteins are subject to beingdigested when administered orally, parenteral administration, i.e.,intravenous, subcutaneous, intramuscular, would ordinarily be used tooptimize absorption.

Monoclonal antibodies of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adaily dosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10milligrams per kilogram per day given in divided doses 1 to 6 times aday or in sustained release form is effective to obtain desired results.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

Cytotoxic drugs which can be conjugated to antibodies and subsequentlyused for in vivo therapy include, but are not limited to, daunorubicin,doxorubicin, methotrexate, and Mitomycin C.

The non-human animals of the invention comprise any animal having atransgenic interruption or alteration of the endogenous gene(s)(knock-out animals) and/or into the genome of which has been introducedone or more transgenes that direct the expression of differentiallyexpressed human PCA3 mRNAs. Also preferred are the introduction ofantisense PCA3 nucleic acids.

Such non-human animals include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, amphibians, reptiles, etc. Preferrednon-human animals are selected from non-human mammalian species ofanimals, most preferably, animals from the rodent family including ratsand mice, most preferably mice.

The transgenic animals of the invention are animals into which has beenintroduced by non-natural means (i.e., by human manipulation), one ormore genes that do not occur naturally in the animal, e.g., foreigngenes, genetically engineered endogenous genes, etc. The non-naturallyintroduced genes, known as transgenes, may be from the same or adifferent species as the animal but not naturally found in the animal inthe configuration and/or at the chromosomal locus conferred by thetransgene. Transgenes may comprise foreign DNA sequences, i.e.,sequences not normally found in the genome of the host animal.Alternatively or additionally, transgenes may comprise endogenous DNAsequences that are abnormal in that they have been rearranged or mutatedin vitro in order to alter the normal in vivo pattern of expression ofthe gene, or to alter or eliminate the biological activity of anendogenous gene product encoded by the gene.

The transgenic non-human animals of the invention are produced byintroducing transgenes into the germline of the non-human animal.Embryonic target cells at various developmental stages are used tointroduce the transgenes of the invention. Different methods are useddepending on the stage of development of the embryonic target cell(s).These methods are well known in the art.

Transgenes may be introduced into non-human animals in order to provideanimal models for human diseases. Transgenes that result in such animalmodels include, e.g., transgenes that encode the differentiallyexpressed PCA3 mRNAs associated with a malignant prostate status (i.e.,prostate cancer) or a non-malignant prostate status.

Having identified a marker sequence in differentially expressed PCA3mRNA and a correlation between the balance of the expression level ofthe differentially expressed PCA3 mRNAs (or protein encoded thereby) andthe malignant or non-malignant prostatic states, the present inventionopens the way to numerous methods, assays and reagents for theprognosis, diagnosis, staging, predisposition and therapy of prostatecancer. In a broad embodiment, the present invention provides the meansto assess prostate cancer by identifying PCA3 mRNA lacking theadditional sequence in accordance with the present invention (or aprotein encoded thereby). Numerous methods, primers, probes, antibodiesand reagents can be used to identify such a nucleic acid molecule (orsuch a protein), as will be clear to the skilled artisan to which thepresent invention pertains.

The present invention is illustrated in further detail by the followingnon-limiting examples.

Example 1 Identification of Differentially Expressed PCA3 mRNAs andCorrelation of their Expression with Prostatic Disease

PCA3-specific PCR primers were developed in order to analyze PCA3expression in different samples. To be able to discern between sequencesamplified from mRNA (messenger RNA) and genomic DNA, these primers weredesigned to span an intron, in occurrence intron 3. As illustrated inFIG. 1, the PCA3 sense primer lies within exon 3 and the PCA3 antisenseprimer within exon 4a. Samples to be analyzed for PCA3 expressionconsisted of frozen tissue chips removed by transurethral resection ofthe prostate (BPH, 4 patients) or frozen prostates obtained by radicalprostatectomy (prostate cancer, 6 patients). Radical prostatectomysamples were processed into frozen sections to specifically selectregions containing prostate cancer cells. RNA was extracted from thefrozen samples using a liquid-phase RNA extraction method (Trizol™).Extracted nucleic acids were subsequently treated with DNase in order todigest genomic DNA. DNase-treated RNA was reverse transcribed into cDNAusing reverse transcriptase and then submitted to PCR-analysis using thePCA3 primers. PCR was performed for 35 cycles with Taq DNA polymerase,amplified material was separated on agarose gels and visualized byethidium bromide staining. As shown in FIG. 2, PCR amplification of PCA3generates two products which can be separated by size and differ inrelative abundance. The smaller amplicon (277 bp) is predominantly orexclusively found in samples from prostate cancer patients (FIG. 2,upper row) whereas the larger amplicon (505 bp) is more prominent insamples from patients with a non-malignant prostatic state (BPH [FIG. 2,lower row]). Pathological examination of patients' biopsies confirmedthe initial diagnosis for each patient except for Patient BPH 1 whichwas found to have prostate cancer.

In order to confirm the origin of the amplified fragments, they wereisolated from the gel and sequenced. Sequences are shown in FIG. 3. Asexpected, the smaller 277 bp fragment proved to correspond to theregions of exons 3 and 4a spanned by the PCA3 PCR primers. The larger505 bp fragment is identical to the smaller fragment except for theherein identified sequence which lies between exon 3 and exon 4a. Ofnote, direct PCR analysis of all samples without reverse transcriptiondid not yield amplified material ruling out the hypothesis that thelarger amplification product originates from genomic DNA.

Thus, PCA3 mRNA is present in at least two distinct forms within thecell, a short form lacking the herein identified additional sequence(hereafter called sequence 2; SEQ ID NO:2) as well as a long form havingthis additional sequence (hereafter called sequence 1; SEQ ID NO:1). Thepresence of the additional sequence in sequence 1 interrupts thepredicted open reading frame coding for the PCA3 protein. The predictedsequence of the protein encoded by this long PCA3 mRNA is shown in FIG.4. As illustrated in FIG. 2, relative expression levels of the two PCA3mRNA sequences vary dependent on the cell type. Prostate cancer cellspredominantly express sequence 2 whereas BPH cells mainly expresssequence 1.

These observations demonstrate that it is possible to discern between amalignant and non-malignant state of a prostate. As well, it is temptingto predict that the relative levels of the two types of PCA3 mRNAs, willenable to discern the benign state from the malignant state.

Example 2 Assessment of the Prostatic State of a Patient Using RT-PCR

Patient samples were obtained and RNA prepared therefrom as commonlyknown. Reverse transcription mixes were prepared as RT follows: 0.2 μgtotal RNA+0.6 μg pdN6 (random hexamer primers)+1.25 mM dNTPs+200 U M-MLVreverse transcriptase in 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂,10 mM DTT. The mixture was incubated 1 hr at 40° C.

4 μl of the RT-reaction of above was mixed in 50 μL of 20 mM Tris-HCl pH8.4, 50 mM KCl, 2.5 mM MgCl₂, 0.5 mM dNTPs, 0.5 μM of each primer and2.5 U Taq DNA polymerase. For PCR analysis, the amplification wascarried out for 35 cycles (1 min each at 94° C., 60° C., 72° C.)followed by a 10 min extension at 72° C. The PCR products were analyzedby conventional agarose gel electrophoresis.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

1. A prostate cancer-classifying composition comprising: (a) prostatecells or a nucleic acid preparation therefrom obtained from a subjecthaving or suspected of having prostate cancer; and (b) at least oneoligonucleotide enabling the determination of whether PCA3 RNA from saidprostate cells or said nucleic acid preparation comprises or lacks anadditional sequence between PCA3 exon 3 and PCA3 exon 4a, saidadditional sequence corresponding essentially to nucleotides 27 to 254of SEQ ID NO: 1, wherein the presence or level of a PCA3 nucleic acidmolecule lacking said additional sequence indicates that said prostatecells comprise prostate cancer cells.
 2. The prostate cancer-classifyingcomposition of claim 1, wherein said determination of whether PCA3 RNAfrom said prostate cells or said nucleic acid preparation comprises orlacks an additional sequence between PCA3 exon 3 and PCA3 exon 4acomprises performing a hybridization and/or amplification reaction. 3.The prostate cancer-classifying composition of claim 2, wherein saidamplification reaction comprises a reaction which is: a nucleic acidsequence-based amplification reaction (NASBA), a polymerase chainreaction (PCR), a transcription-based amplification reaction, a stranddisplacement amplification (SDA), a ligase chain reaction (LCR), or a Q3replicase reaction.
 4. The prostate cancer-classifying composition ofclaim 1, wherein said oligonucleotide hybridizes specifically to: (a)PCA3 exon 3; (b) PCA3 exon 4a; (c) the PCA3 exon 3-exon 4a junction; (d)the PCA3 exon 3-intron 3 junction; or (e) the PCA3 intron 3-exon 4ajunction.
 5. The prostate cancer-classifying composition of claim 1,wherein said oligonucleotide hybridizes specifically to a PCA3 exon3-exon 4a junction as defined by nucleotide positions 26 and 27 of SEQID NO:
 2. 6. The prostate cancer-classifying composition of claim 1,wherein said oligonucleotide is at least 10 nucleotides in length. 7.The prostate cancer-classifying composition of claim 1, wherein saidoligonucleotide is at least 12 nucleotides in length.
 8. The prostatecancer-classifying composition of claim 1, wherein said oligonucleotideis at least 15 nucleotides in length.
 9. The prostate cancer-classifyingcomposition of claim 1, wherein said oligonucleotide is at least 20nucleotides in length.
 10. The prostate cancer-classifying compositionof claim 1, wherein said oligonucleotide is at least 18 to about 50nucleotides in length.
 11. The prostate cancer-classifying compositionof claim 1, wherein said oligonucleotide is at least 20 to about 35nucleotides in length.
 12. The prostate cancer-classifying compositionof claim 1, wherein said oligonucleotide is detectably labeled.
 13. Theprostate cancer-classifying composition of claim 1, wherein saidoligonucleotide hybridizes under high stringency conditions to at least10 consecutive nucleotides of a sequence comprising nucleotides 26 and27 of SEQ ID NO:
 2. 14. The prostate cancer-classifying composition ofclaim 1, wherein said oligonucleotide hybridizes under high stringencyconditions to at least 15 consecutive nucleotides of a sequencecomprising nucleotides 26 and 27 of SEQ ID NO:
 2. 15. The prostatecancer-classifying composition of claim 1, wherein said oligonucleotidehybridizes under high stringency conditions to at least 20 consecutivenucleotides of a sequence comprising nucleotides 26 and 27 of SEQ ID NO:2.
 16. The prostate cancer-classifying composition of claim 1, whereinsaid high stringency conditions comprise a hybridization at 65° C. in5×SSC, 5×Denhardt's solution, 1% SDS, and 100 μg/mL denatured carrierDNA.
 17. The prostate cancer-classifying composition of claim 1 furthercomprising a DNA polymerase.
 18. The prostate cancer-classifyingcomposition of claim 1, wherein said composition enables thedetermination of the malignancy status of said subject having orsuspected of having prostate cancer.
 19. The prostate cancer-classifyingcomposition of claim 2, wherein said composition enables thedetermination of the malignancy status of said subject having orsuspected of having prostate cancer.
 20. The prostate cancer-classifyingcomposition of claim 5, wherein said composition enables thedetermination of the malignancy status of said subject having orsuspected of having prostate cancer.